Approximately 30% of the mammalian proteome is synthesized into the endoplasmic reticulum (ER) and enters the secretory pathway. The folding of these proteins is a complicated process due to the crowded environment of the ER. Protein misfolding in the ER is common and occurs continually because of genetic mutations, errors during transcription/translation, or conditions of stress. Over 70 diseases are related to protein misfolding or defects in protein homeostasis. Misfolded proteins within the ER are dislocated back into the cytosol across the ER membrane for degradation by the proteasome. This quality control process called ER-associated degradation (ERAD) requires a complex system of proteins in the ER lumen, membrane, and cytosol. The molecular mechanisms governing this process are unclear. ERAD machinery is hypothesized to be modular around membrane embedded E3 ubiquitin ligases. Hrd1 is one such E3 ubiquitin ligase functioning in ERAD with several known binding partners. However, most studies to date have examined the composition of mammalian Hrd1 complexes in the context of protein overexpression, which can result in artifactual protein- protein interactions. Therefore, genome editing techniques were utilized to construct cell lines permitting the biochemical analysis of Hrd1 complexes expressed at endogenous levels. The overall goal of this research is to comprehensively dissect the architecture of Hrd1 complexes using this state-of-the-art approach. Specific aim #1 of this proposal combines biochemical fractionation and mass spectrometry (MS) to define components of native Hrd1 complexes at steady-state. In the second aim, absolute quantitation MS (AQUA-MS) will be used to reveal the stoichiometry of components within identified Hrd1 complexes. These experiments will provide an unprecedented level of detail in the architecture of ERAD complexes and yield new insight into the function of this protein quality control system. Specific aim #3 explores the dynamic adaption of these complexes to cellular perturbations. These studies involve applying the approaches and workflow developed in aims #1 and #2 in distinct cellular contexts, including stress conditions and the expression of disease-related ERAD substrates. Together, the research proposed will significantly advance our understanding of the functional networks involved in ERAD.